Our research interests are to map and isolate novel cancer-causing genes, to clarify their normal functions and their relationship to environmental carcinogenesis. To facilitate this process, we have developed human gene targeting system using recombination proficient chicken DT40/human cell hybrids. Any human gene or chromosome in chicken DT40 cell can be targeted and modified at high frequency. We have been developing the following three applications of this system; 1) a novel yeast artificial chromosome (YAC) cloning method which allows one to isolate a specific region of DNA segment by gene targeting; 2) a system which allows one to assess the functions of a specific gene by disruption of the gene (gene knock-out); 3) a system which allows one to create a truncated transferable chromosome at specific gene locus by targeted insertion of a telomere sequence. One of our research projects has been directed toward identifying a novel cellular senescence gene on human chromosome 1 whose introduction into the human osteosarcoma cell line, TE85, is supposed to induce growth arrest. To localize a gene to a clonable size of the chromosomal region, we generated mouse A9 hybrid cells containing a truncated chromosome 1 at various sites of this chromosome by a random insertion of a telomere sequence. Surprisingly, transfers of an intact chromosome 1 as well as any of the truncated chromosomes 1 into TE85 did not induce senescence, suggesting that chromosome 1 does not carry a senescence-inducing gene. We are currently transferring other human chromosomes including chromosomes 1, 2, 3, and 6 into TE85 to identify the human chromosome that carries senescence activity for TE85 cells. We are constructing a minicromosome, which can carry a single copy of BAC or YAC clone, to facilitate gene mapping as well as functional analysis of a single gene. A minichromosome will be generated from chromosome 11 in DT40 cells by targeted insertion of a telomere sequence into the loci close to a centromere of this chromosome. To determine the functions of the human mismatch repair gene, hMSH3, we have transferred a human chromosome 5 carrying a wild type hMSH3 into the hMSH3-defective human colon cell line, HCT116.